Design, synthesis and tumor cell growth inhibitory activity of 3-nitro-2H-cheromene derivatives as histone deacetylaes inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.05.062

As a continuous research for the discovery of coumarin-based targeted anticancer agents, we designed and synthesized a series of novel histone deacetylases (HDAC) inhibitors using the 8-ethoxy-3-nitro-2H-chromene as the surface binding or cap group, linear dicarboxylic acid or ω-amino acid moiety with different length as the linking motif, ortho-aminoanilides, amides or α-aminoamides as the zinc binding group and the internal cavity motifs. Most of these 3-nitro-2H-chromene derivatives exhibited good growth inhibitory activity against K562, A549, MCF-7, PC3 and Hela cells and were more potent than the reference drug SAHA and MS-275. At the concentration of 10?μM, the ortho-aminoanilide series and the D-Phe derived α-aminoamide derivatives 16a and 16b displayed more potent activity toward HADC1 over HADC2, and only moderate to weak activity over HADC6. In contrast, the amide ZBG analogues, 12a and 12b, 14 and 15, were only moderate HDAC6 inhibitors, but more selective over HDAC1 and HDAC2. The ortho-aminoanilides 9b, 9c, 10b, 10c, 11b, and the α-aminoamides 16a and 16b were potent HADC1 inhibitors with the IC₅₀ values in the nanomolar ranges. The ortho-aminoanilides 10b and10c with a phenyl internal cavity motif were more potent than MS-275 as HADC1 inhibitors and more selective over HADC2.

Full text of DOI:10.1016/j.bmc.2017.05.062

Accepted Manuscript
Design, synthesis and tumor cell growth inhibitory activity of 3-nitro-2H-cher-
omene derivatives as histone deacetylaes inhibitors
Shuai Tan, Feng He, Tingting Kong, Jingde Wu, Zhaopeng Liu
PII:
S0968-0896(17)30832-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.062
BMC 13781
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 April 2017
27 May 2017
31 May 2017
Please cite this article as: Tan, S., He, F., Kong, T., Wu, J., Liu, Z., Design, synthesis and tumor cell growth inhibitory
activity of 3-nitro-2H-cheromene derivatives as histone deacetylaes inhibitors, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.062
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Design, synthesis and tumor cell growth inhibitory activity
of 3-nitro-2H-cheromene derivatives as histone deacetylaes
inhibitors
†
Shuai Tan , Feng He , Tingting Kong, Jingde Wu* and Zhaopeng Liu*
†
Institute of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School
of Pharmaceutical Sciences, Shandong University, Jinan 250012, China
*Corresponding authors
Zhaopeng Liu, Tel. +86-531-88382006; Fax: +86-531-88382548; E-mail: liuzhaop@sdu.edu.cn；
Jingde Wu, Tel. +86-531-88382006; Fax: +86-531-88382548; wujingde70@sdu.edu.cn
†
These two authors contributed equally to this paper.
Abstract
As a continuous research for the discovery of coumarin-based targeted anticancer agents, we
designed and synthesized a series of novel histone deacetylases (HDAC) inhibitors using the
8-ethoxy-3-nitro-2H-chromene as the surface binding or cap group, linear dicarboxylic acid or ω-amino
acid moiety with different length as the linking motif, ortho-aminoanilides, amides or α-aminoamides
as the zinc binding group and the internal cavity motifs. Most of these 3-nitro-2H-chromene derivatives
exhibited good growth inhibitory activity against K562, A549, MCF-7, PC3 and Hela cells and were
more potent than the reference drug SAHA and MS-275. At the concentration of 10 µM, the
ortho-aminoanilide series and the D-Phe derived α-aminoamide derivatives 16a and 16b displayed
more potent activity toward HADC1 over HADC2, and only moderate to weak activity over HADC6.
In contrast, the amide ZBG analogues, 12a and 12b, 14 and 15, were only moderate HDAC6 inhibitors,
but more selective over HDAC1 and HDAC2. The ortho-aminoanilides 9b, 9c, 10b, 10c, 11b, and the
α-aminoamides 16a and 16b were potent HADC1 inhibitors with the IC50 values in the nanomolar
ranges. The ortho-aminoanilides 10b and10c with a phenyl internal cavity motif were more potent than
MS-275 as HADC1 inhibitors and more selective over HADC2.
Keywords：HDACs, HDAC inhibitors, 3-nitro-2H-chromenes, coumarins, anticancer agents,
Zinc-dependent hydrolases, isoform selective

1
. Introduction
As a family of hydrolases that remove acetyl groups from lysine residues, histone deacetylases
(HDACs) play very important roles in the regulation of multiple processes, from gene expression to
protein activity [1, 2]. The dysregulation of HDACs is implicated in many diseases, such as cancer,
cellular metabolism disorders, neurological disorders, and inflammation. To date, 18 HDACs have been
identified in mammals. According to the differences in their sequence identity and catalytic activity,
HDACs can be divided into four classes. Class I includes HDAC1, 2, 3 and 8, which are mainly located
in the nucleus. Class II includes HDAC4, 5, 6, 7, 9, and 10, which are tissue specific and shuttle
+
between the cytoplasm and the nucleus [3, 4]. Class III, also called sirtuins, are NAD -dependent
2
+
HDACs. Like classes I and II, class IV is metal (Zn ) dependent and has only one member, HDAC11,
which is mainly located in the nucleus. The active site of zinc-dependent HDACs include four
prominent binding domains: the surface binding domain, the hydrophobic channel, the catalytic zinc
binding domain, and the adjacent internal cavity. The pharmacophore model of HDACs inhibitors
(HDACi) can also be divided into four groups: a surface binding or cap group (Cap), a hydrocarbon
linking motif (Linker), a zinc binding group (ZBG) and an internal cavity motif correspondingly [5, 6].
A variety of aromatic rings, heterocyclic rings and macrocycles can serve as the cap group. The linker
motif is usually an alkyl chain or a substituted aromatic ring. The most common ZBG are hydroxamic
acid and ortho-aminoanilide. The internal cavity motif can be a variety of aromatic or heterocyclic
rings [7, 8]. Some licensed HDACi, including SAHA, Romidepsin, Belinostat, Panobinostat and
Chidamide, not only confirmed the safety and efficacy of HDACi as anticancer drugs, but also attracted
more attentions for the discovery novel selective HDACi for the treatment of a variety of diseases.
Coumarins are fused benzene and pyrone ring systems that exist abundantly in nature. Both
natural and synthetic coumarin derivatives possess a broad spectrum of biological activities, including
antioxidant, anti-inflammatory, anticancer, antiviral, antihyperlipidemic, MAO-B inhibitory and
antimicrobial activities, among many others [9, 10]. In recent years, the synthesis of coumarins and
their derivatives has attracted considerable attention in the development of novel anticancer agents [11,
12, 13]. A coumarin derivative, 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161), was
discovered as a PI3K/Akt signal pathway inhibitor through high-throughput screening that displayed
potent antileukemia and antimyeloma activity with minimal toxicity [14]. Structural simplification and

modification of S14161 by us led to the identification of 6-bromo-8-ethoxy-3-nitro-2H-chromene
BENC-511) that exhibited more potent anti-proliferative activities against a panel of cell lines [15].
(
Compared with S14161, BENC-511 was more potent in blocking the AKT phosphorylation and
inducing cancer cell apoptosis, and demonstrated potent oral activity against myeloma in vivo and great
therapeutic efficacy in a PC3-derived prostate cancer model in nude mice [16, 17]. However,
BENC-511 showed little inhibitory effects on the PI3Ks and its molecular targets have not been
identified. To develop targeted anticancer drugs, we herein report the design, synthesis and biological
evaluation of a series of novel 3-nitro-2-chromene derivatives as potent histone deacetylaes inhibitors.
In these HDACi, the 8-ethoxy-3-nitro-2H-chromene served as the Cap group, linear dicarboxylic acid
or ω-amino acid moiety acted as the Linker, ortho-aminoanilides, amides or α-aminoamides behaved as
the ZBGs and the internal cavity motifs (Figure 1).
NH2
H
N
H
N
O N
2
( )n
NO2
O
O
O
OEt
R
O
9a–c; 10a–c; 11a–c
OEt
F
H
N
H
N
S14161
O N
2
( )n
Bn
O
O
O
O
Br
NO2
OEt
12a–c
O
H
N
O
O N
2
( )5 N
H
R
OEt
O
BENC-511
OEt
OEt
1
4; 15
O
H
N
Cap group
Linker
Zinc binding group (ZBG)
Internal cavity motif
O N
2
( )n
N
Ph
H
O
NH2
O
1
6a; 16b
Figure 1. The design of 3-nitro-2H-chromene derivatives as HDACi.
2
. Results and discussion
2.1. Chemistry
In the design of 3-nitro-2-chromene based histone deacetylaes inhibitors, the
-ethoxy-3-nitro-2H-chromene was designed as the Cap moiety, an amino group was introduced at the
8

6
-position to replace the 6-bromo in BENC-511 through which the dicarboxylic acid or ω-amino acid
linker was connected by an amide bond. As shown in Scheme 1, the nitration of
-ethoxy-2-hydroxybenzaldehyde in glacial acetic acid generated the 6-nitro derivative 1 in 71% yield.
The protection of both the hydroxyl and aldehyde group was achieved in high yield (92%) by the
treatment of 1 with Ac O in the presence of a catalytic amount of concentrated sulfuric acid. The Pd/C
catalytic hydrogenation of 2 provided the 6-amino derivative, which was unstable and reacted
immediately with (Boc) O to protect the resulting amino group, followed by the removal of the acetyl
3
2
2
protective groups in refluxing aqueous NaOH solution to give the salicylaldehyde derivative 3 in 76%
yield (3 steps). The reaction of salicylaldehyde 3 with 2-nitroethanol in the present of 200% mol of
phthalic anhydride and using dibutylamine as a base in toluene under reflux conditions gave the
corresponding 6-BocNH-8-ethoxy-3-nitro-2H-chromene 4 in 50% yield.
CHO
OH
O N
CHO
OH
O N
CH(OAc)2
OAc
2
2
a
b
c
7
1%
92%
76%
OEt
OEt
OEt
1
2
BocHN
CHO
OH
BocHN
NO2
d
50%
O
OEt
OEt
3
4
Scheme 1. Synthesis of the 6-BocNH-8-ethoxy-3-nitro-2H-chromene 4. Reagents and conditions:
o
(
a) HNO , AcOH, 0 C to rt, 4 h; (b) Ac O, H SO , 2 h; (c) i) 10%Pd/C, H , THF, 25 h; ii) (Boc) O,
3
2
2
4
2
2
H O, 30 h; iii) NaOH, reflux 8 h; (d) phthalic anhydride, n-Bu NH, 2-nitroethanol, toluene, reflux, 18 h.
2
2
To examine the influences of linker length on the HDAC inhibitory activity, linear dicarboxylic
acids of 6-, 7- and 8-carbon lengths were applied. In these dicarboxylic acids derived series, the
ortho-aminoanilide (9−11) and the amide (12) were used as the ZBGs. In the ortho-aminoanilide series,
the phenyl (10a−c) and 5-methylthiophen-2-yl (11a−c) acted as the internal cavity motifs that were
expected to improve both the HDAC inhibitory activity and the isoform selectivity [18, 19]. Besides
the linear dicarboxylic acid, the 5-aminopentanoic acid or 6-aminohexanoic acid was used as the linker
motif, and the derived ω-amides (14, 15) or the α-amino amides (16a, 16b) behaved as the ZBGs. The
synthesis of these 8-ethoxy-3-nitro-2H-chromene based HDACi was shown in Scheme 2.

Treatment of 4 with 20% trifluoroacetic acid (TFA) solution in dichloromethane removed the Boc
protective group to give the free amine, which was coupled with adipic acid, heptanedioic acid and
octanedioic acid, in the presence of HATU and triethylamine in DMF, to afford the acid derivatives 5a,
5b and 5c in 73%, 74% and 79% yield, respectively. Coupling the resulting free amine with the
5-BocNH-pentanoic acid or 6-BocNH-hexanoic acid under the same conditions resulted in 13a and 13b
in 92% and 89% yield. The reaction of the acids, 5a, 5b, and 5c, with tert-butyl
-aminophenylcarbamate, in the presence of HATU and triethylamine in DMF, provided the amides 6a
61%), 6b (61%) and 6c (60%). By the same method, the reaction of 5a, 5b, or 5c with tert-butyl
2
(

3-amino-4-(1,1'-biphenyl)-4-yl)carbamate,
tert-butyl
3-amino-4-(5-methylthiophen-2-yl)phenyl)carbamate or phenylethylamine, respectively, generated the
amides 7a (47%), 7b (64%), 7c (66%), 8a (57%), 8b (58%), 8c (74%), or 12a (67%), 12b (62%), 12c
65%). Treatment of 6a−c, 7a−c, and 8a−c with TFA, respectively, produced the corresponding
diamides 9a (65%), 9b (56%), 9c (51%), 10a (96%), 10b (96%), 10c (94%), or 11a (89%), 12b (88%),
2c (88%). After the removal of the Boc protective group of 13a or 13b with TFA, the resulting amine
(
1
from 13b was reacted with phenylacetic acid or 3-phenylpropanoic acid, in the presence of HATU and
triethylamine in DMF, to give the ω-amides 14 (95%) and 15 (93%), while the reaction of the amines
derived from both 13a and 13b with the D-Boc-Phe, followed by the deprotection of the Boc group
with TFA in CH
.2. In vitro cell growth inhibitory activity
The newly synthesized 8-ethoxy-3-nitro-2H-chromene based HDACi were evaluated for their
2 2
Cl , provided the corresponding ω-(α)-aminoamide 16a (51%) and 16b (48%).
2
antiproliferative activity against five human cancer cell lines, K562 (human leukemia), A549 (human
lung carcinoma), MCF-7 (human breast adenocarcinoma), PC3 (human prostate cancinoma), and HeLa
(human cervical carcinoma), by the conventional MTT assay, using the clinical drug SAHA and the
typical benzamide HDAC inhibitor MS-275 as the reference drugs [20]. The results are shown in Table
1.
Like SAHA and MS-275, these 8-ethoxy-3-nitro-2H-chromene derivatives showed different
sensitivity towards K562, A549, MCF-7, PC3 and HeLa cells, and most of them were more sensitive to
K562 cells than the others, and they exhibited high potency against K562 with GI50 values ranging
from 1.53 to 6.04 µM. All of them were more potent than MS-275 and most of them were highly active
or comparable to SAHA against K562 cells. Except for a few compounds (9b, 11b), all the others were
more active or at least comparable to the reference drugs SAHA and MS-275, toward the A549, MCF-7
and PC3 cell lines. Though they were relatively less sensitive towards the HeLa cells, compounds 12a,
12b, 14 and 15 were more potent than SAHA and MS-275, while compounds 9a, 9b, 10b, 10c and 11a
displayed comparable potency.
In general, the linker, the kind of ZBGs and the internal cavity motifs affected the antiproliferative
activity, but in different degrees, depending on the cell types. Among the ortho-aminoanilide series
(9~11a−c), the compounds with a phenyl (10a−c) or 5-methylthiophen-2-yl (11a−c) internal cavity
motif, in most cases, showed increased potency against the K562, A549, MCF-7 and PC3 cell lines in

comparison with their counterparts 9a−c. Compounds 12a and 12b with the amide ZBG groups
exhibited relatively higher activity than the ortho-aminoanilide series having the same linear
dicarboxylic acid derived linkers, especially, they were potent toward all the five tumor cell lines tested.
In contrast, their counterpart 12c with one- or two-carbon elongation in the linker displayed much
reduced activity than 12a and 12b. The 2-phenylacetamide 14 and the 3-phenylpropanamide 15 with
the 6-aminohexanoic acid derived linkers displayed similar potency as that of 12a and 12b toward the
five tumor cell lines, however, the D-Phe derivatives 16a and 16b ere inactive for the HeLa cell line
and exhibited a slightly reduced potency than 14 and 15 against the K562, A549, MCF-7 and PC3 cell
lines.
Table 1: Antiproliferative activity and HDACs inhibitory potency in vitro
a
b
GI50 (µM)
% inhibition of HDACs
Compd.
K562 A549 MCF-7 PC-3 HeLa HDAC1 HDAC2 HDAC6
9
9
a
3.68
9.63 17.32 18.91 13.07
89
95
91
96
93
59
81
72
68
57
52
25
28
29
b
3.96 12.18 19.45 > 20 14.93
3.36 11.67 13.47 8.63 > 20
9
c
100
95
1
0a
0b
5.27
3.21
2.00
7.46 19.93 14.59 > 20
1
4.86
5.40
7.03
9.37
7.08 19.79
10.74 16.24
100
94
1
1
0c
1a
2.94 13.75 7.91
13.48 18.35
11.38 > 20
6.36 > 20
66
90
83
5
32
62
55
3
8
1
1b
2.92
3.11
2.90
3.47
5.07
5.83
6.42
5.32
> 20
5.33
2.82
6.12
36
19
54
40
14
51
43
1
1
1c
2a
6.46
7.09
9.33
6.08
1
2b
2c
11
23
5
0
1
4.12 18.21 19.11 10.94 > 20
8
1
4
5
1.53
3.32
3.56
4.80
5.23
5.95
4.17
5.96
8.72
13.15 5.80
7.17 6.74
11.17 > 20
0
1
0
8
1
6a
99
100
95
90
100
98
60
100
3
SAHA 3.66 19.45 17.90 18.83 19.21
MS-275 9.32 18.37 19.26 19.09 19.24
a
b
three replicates with SD values <20% of the mean; at 10 µM.
2.3. In vitro HDACs inhibitory activity
Selective HDAC inhibitors would provide powerful chemical tools to dissect the individual
functions of the HDAC isoforms, in addition to providing lead antitumor drug candidates. To assess the
isoform selectivity of the 8-ethoxy-3-nitro-2H-chromene analogues, HDAC1 and HDAC2 representing
class I and HDAC6 representing class II were used in the in vitro inhibitory assay. At the concentration

of 10 µM, these 8-ethoxy-3-nitro-2H-chromene derivatives exhibited quite different inhibitory rates
Table 1), depending on mainly on the ZBG type and internal cavity group. The ortho-aminoanilides
a−c with no internal cavity group showed good inhibiting rates for HDAC1 and HDAC2, and
moderate activity for HDAC6, while the ortho-aminoanilides with phenyl (10a−c) or
-methylthiophen-2-yl (11a−c) internal cavity group exhibited comparable potency for HDAC1, but
(
9
a
5
reduced activity for HDAC2 and HDAC6. The linker length had slight influences on the activity of
these ortho-aminoanilides, and in general, the heptanedioic acid derived linker was preferred. In
comparison with the ortho-aminoanilide series, the weak amide ZGB derivative 12a and 12b, 14 and
15, showed some selectivity toward HDAC6, since they had moderate inhibitory activity for HDAC6,
but little inhibition against HDAC1 and HDAC2. It is of notice that the D-Phe derived analogues 16a
and 16b showed similar potency as that as the ortho-aminoanilides against HDAC1, 2 and HADC6. It
is apparent that the amino group at the α-position of the amide can contribute to the binding with the
2+
Zn .
The IC50 values for those compounds that showed relatively good inhibitory activity toward
HADC1, 2 at 10 µM had been determined (Table 2). The ortho-aminoanilides 9b, 9c, 10b, 10c, 11b,
and the α-aminoamides 16a and 16b were potent HADC1 inhibitors with the IC50 values in the
nanomolar ranges. The ortho-aminoanilides 10b and10c with a phenyl internal cavity motif were more
potent than MS-275 and more selective of HADC1 to HADC2.
Table 2: The IC50 values of the selected compounds for HDAC1 and HDAC2
IC50 (nM)
HDACs
9
b
9c
10b
128
659
10c
179
827
11b
366
16a
742
16b
448
MS275
271
SAHA
HDAC1
HDAC2
288
759
232
439
9
1788
2915
1294
505
17
3
. Conclusion
In summary, a series of 8-ethoxy-3-nitro-2H-chromene based HDACIs were designed, synthesized
and evaluated for their inhibitory activity against K562, A549, MCF-7, PC3 and HeLa cell lines and
HDAC1, 2 and HDAC6. Most of these coumarin derivatives displayed good antiproliferative activity
and were more potent toward the five tumor cell lines rested than the reference drug SAHA and

MS-275. The ortho-aminoanilide and the D-Phe derived α-amnoamide derivatives (16a and 16b)
displayed more potent activity toward HADC1 over HADC2, and only moderate to weak activity over
HADC6. In contrast, the amide ZBG analogues (12a and 12b, 14 and 15) were only moderate HDAC6
inhibitors, but more selective over HDAC1 and HDAC2. The ortho-aminoanilides 9b, 9c, 10b, 10c,
11b, and the α-aminoamides 16a and 16b were potent HADC1 inhibitors with the IC50 values in the
nanomolar ranges. The ortho-aminoanilides 10b and10c with a phenyl internal cavity motif were more
potent than MS-275 as HADC1 inhibitors and more selective over HADC2. In comparison with the
amide ZBG derivatives, the D-Phe derived analogues were more potent HDAC inhibitors. The
application of amino acids as both ZBG and internal cavity motifs will be hopeful in the discovery
potent and selective HDAC inhibitors.
4
. Experiment sections
4.1. Chemmistry
Melting points were determined on an X-6 micro-melting point apparatus (Beijing Tech. Co., Ltd,
1 13
China, Beijing) and are uncorrected. H and C NMR spectra were recorded on a AVANCE 600
(Bruker, Switzerland) instrument with a 600/150 MHz operating frequency at room temperature for
CDCl
3
solutions, unless otherwise stated. All chemical shifts were reported as δ values (ppm) relative
to Me
4
Si (0.00 ppm) as internal standards. Electrospray-ionization mass spectrometry (ESI-MS) was
performed on an API 4000 (AB SCIEX, USA) instrument. Column chromatography was performed on
silica gel (200−300 mesh). Independent analysis of purity for synthetic products was achieved using
analytical HPLC system (Agilent 1100 series, Agilent Technologies, Germany). All the compounds for
the biological assay are of high purities (>98.5%). All the reactions involving oxygen- or
2
moisture-sensitive compounds were carried out under a dry N atmosphere. Unless otherwise noted,
reagents were added by syringe. THF was distilled from sodium/benzophenone immediately prior to
use.
4.1.1. Synthesis of 3-ethoxy-2-hydroxy-5-nitrobenzaldehyde (1)
To a solution of 3-ethoxy-2-hydroxybenzaldehyde (6.7 g, 40.3 mmol) in glacial acetic acid (20 mL)
was added the mixture solution of nitric acid (4.85 mL) and glacial acetic acid (20 mL) at 0 °C. After
stirring at room temperature for 4 h, the reaction mixture was poured into ice water (150 mL). The
resulting precipitate was filtered, washed with a small amount of ice water. The crude product was

recrystallized from EtOAc and petroleum (1:2) to give 6.0 g of 1 as a yellow solid, yield 71%, mp.
1
59–161 °C. H NMR (600 MHz, DMSO-d
1
6
) δ 11.54 (s, 1H), 10.33 (s, 1H), 8.11 (d, 1H, J = 2.4Hz),
7.91 (d, 1H, J = 2.4 Hz), 4.26 (q, 2H, J = 7.2 Hz),1.41 (t, 3H, J = 6.6 Hz); HRESI-MS calcd. for
+
[M+H] 212.0153, found 212.0149.
C H
9
10NO
5
4.1.2 Synthesis of (2-acetoxy-3-ethoxy-5-nitrophenyl)methylene diacetate (2)
To a solution of 1 (4.1 g, 19.4 mmol) in Ac O (100 mL) was added 6 drops of concentrated
2
sulfuric acid. After stirring at room temperature for 2 h, the reaction mixture was poured into ice and
water (500 mL). After stirring for 40 min, the resulting precipitate was filtered, washed with a small
1
amount of icy water, dried, to give 6.20 g of 2 as a white solid, yield 92%, mp. 113–114 °C. H NMR
(600 MHz, DMSO-d
6
) δ 8.00 (dd, 2H, J = 1.8, 6.0 Hz), 7.76 (s, 1H), 7.91 (d, 1H, J = 2.4 Hz), 4.23 (q,
2H, J = 7.2 Hz), 2.32 (s, 1H), 2.11 (s, 6H), 1.31 (t, 3H, J = 6.6 Hz); HRESI-MS calcd. for C15H18NO
9
+
[
M+H] 356.0976, found 356.0978.
.1.3. Synthesis of tert-butyl (3-ethoxy-5-formyl-4-hydroxyphenyl)carbamate (3)
A mixture of 2 (3.0 g, 8.45 mmol) and 10% Pd/C (0.3 g) in THF (10 mL) was stirred under H
atmosphere at room temperature for 25 h. The reaction mixture was filtered, and the filtrate was
evaporated. To the resulting residue, water (20 mL) and Boc O (4.4 g，40.3 mmol) were added. After
stirring at room temperature for 12 h, additional Boc O (2.2 g，80.6 mmol) was added, and the stirring
was continued for 18 h. EtOAc (30 mL) was added. The water phase was extracted with EtOAc (30
mL). The organic layer was washed with water and brine, dried over Na SO , filtered, and concentrated
4
2
2
2
2
4
under reduced pressure. The resulting residue was dissolved in THF (130 mL), and a 10 M aqueous
NaOH solution (15 mL) was added. After refluxing for 8 h, 10% aqueous citric acid was added to
adjust the pH to 5. After the extraction with EtOAc, the organic phase was washed with brine, dried
2 4
over Na SO , filtered, and concentrated. The resulting residue was purified by column chromatography
on silica gel (petroleum/EtOAc: 5/1) to afford 1.81 g of 3 as a light yellow solid, yield 76%, mp.
1
25–126 °C. H NMR (600 MHz, DMSO-d
1
6
) δ 10.23 (s, 1H), 9.75 (s, 1H), 9.26 (s, 1H), 7.35 (s, 2H),
4.03 (q, 2H, J = 7.2 Hz), 1.47 (s, 9H), 1.37 (t, 3H, J = 6.6 Hz); HRESI-MS calcd. for C14H20NO
5
+
M+H] 282.1336, found 282.1342.
[
4
.1.4. Synthesis of tert-butyl (8-ethoxy-3-nitro-2H-chromen-6-yl)carbamate (4)
To a solution of 3 (4.68 g, 17.79 mmol) in anhydrous toluene (150 mL) was added phthalic
anhydride (4.93 g, 36.05 mmol) and di-n-butylamine (1.60 mL, 9.5 mmol). The mixture was refluxed

with a Dean and Stark apparatus to remove the water. A total of 2.10 mL of 2-nitroethanol (29.3 mmol)
was added portionwise within 12 h. After refluxing for another 36 h, EtOAc (150 mL) was added. The
4
organic layer was washed with water, brine, and dried over anhydrous NaSO , filtered, and
concentrated under reduced pressure. The residue was purified by column chromatography on silica gel
1
EtOAc/hexane: 1/20) to give 2.8 g of 4 as a pale red solid, yield 50%, mp. 203–204 °C. H NMR (600
(
MHz, DMSO-d
s, 9H), 1.33 (t, 3H, J = 7.2 Hz); HRESI-MS calcd. for C16
.1.5. Synthesis of 6-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-6-oxohexanoic acid (5a)
To a solution of 4 (130 mg, 0.39 mmol) in dichloromethane (1.6 mL) was added TFA (0.4 mL).
6
) δ 9.33 (s, 1H), 8.00 (s, 1H), 7.22 (s, 1H), 5.17 (s, 2H), 3.98 (q, 2H, J = 6.6 Hz), 1.47
+
(
21 2 6
H N O [M+H] 337.1394, found 337.1390.
4
After stirring at room temperature for 2 h, the mixture was concentrated under reduce pressure. The
residue was dissolved in anhydrous DMF, and adipic acid (169 mg, 1.20 mmol), HATU (163 mg, 0.43
mmol) and triethylamine (0.43 mL, 3.09 mmol) were added. After stirring at room temperature under
nitrogen atmosphere for 12 h, the reaction mixture was poured into 20 mL brine. The precipitate was
filtered, washed with a small amount of water, and then dissolved in 40 mL ethyl acetate and 20 mL
methanol. The insoluble residues were filtered and the filtrate was concentrated under reduced pressure.
Recrystallization from EtOAc and hexane (1:2) afforded 103 mg of 5a as an orange-red solid, yield
1
3%, mp. 181–183 °C. H NMR (600 MHz, DMSO-d
7
6
) δ 12.02 (s, 1H), 9.90 (s, 1H), 8.02 (s, 1H), 7.39
(d, J = 1.4 Hz, 1H), 7.30 (d, J = 1.6 Hz, 1H), 5.18 (s, 2H), 4.00 (q, J = 6.8 Hz, 3H), 2.28 (t, J = 7.1 Hz,
1
3
2
H), 2.23 (t, J = 7.1 Hz, 2H), 1.63–1.48 (m, 4H), 1.33 (t, J = 6.9 Hz, 3H); C NMR (150 MHz,
DMSO-d
6
) δ 174.8, 171.4, 147.0, 140.4, 139.8, 134.5, 129.9, 119.3, 112.7, 110.1, 64.6, 62.8, 36.5, 33.9,
+
2
21 2 7
5.1, 24.6, 15.1; HRESI-MS calcd. for C17H N O [M+H] 365.1343, found 365.1336.
4.1.6. 7-((8-Ethoxy-3-nitro-2H-chromen-6-yl)amino)-7-oxoheptanoic acid (5b)
Following the same procedures described for 5a, intermediate 5b was prepared from 4 (130 mg,
.39 mmol), TFA (0.4 mL); heptanedioic acid (192 mg, 1.20 mmol), HATU (163 mg, 0.43 mmol) and
0
triethylamine (0.43 mL, 3.09 mmol), in 74% yield (110 mg), as an orange-red solid, mp. 186–187 °C.
1
H NMR (600 MHz, DMSO-d
6
) δ 11.99 (s, 1H), 9.89 (s, 1H), 8.02 (s, 1H), 7.39 (d, J = 2.2 Hz, 1H),
7
2
.29 (d, J = 2.2 Hz, 1H), 5.18 (d, J = 0.7 Hz, 2H), 4.00 (q, J = 6.9 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H),
1
3
.20 (t, J = 7.3 Hz, 2H), 1.60–1.47 (m, 4H), 1.33 (t, J = 7.0 Hz, 3H), 1.30–1.24 (m, 2H); C NMR (150
MHz, DMSO-d
6
) δ 174.5, 171.1, 146.6, 139.9, 139.3, 134.1, 129.5, 118.8, 112.3, 109.6, 64.1, 62.3,
+
6.2, 33.6, 28.2, 24.8, 24.3, 14.7; HRESI-MS calcd. for C18H N O [M+H] 379.1500, found
3
23 2 7

379.1498.
4
.1.7. 8-((8-Ethoxy-3-nitro-2H-chromen-6-yl)amino)-8-oxooctanoic acid (5c)
Following the same procedures described for 5a, intermediate 5c was prepared from 4 (120 mg,
0.34 mmol), TFA (0.4 mL); octanedioic acid (186 mg, 1.06 mmol), HATU (137 mg, 0.36 mmol) and
1
triethylamine (0.4 mL, 2.85 mmol), in 79% yield (106 mg), as an orange-red solid, mp. 196–198 °C. H
NMR (600 MHz, DMSO-d
6
) δ 11.97 (s, 1H), 9.89 (s, 1H), 8.03 (s, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.31
(s, 1H), 5.19 (s, 2H), 4.01 (t, J = 10.4 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.20 (t, J = 7.3 Hz, 2H),
1
3
1
1
2
4
6
.62–1.47 (m, 4H), 1.34 (t, J = 7.0 Hz, 4H), 1.36–1.28 (m, 4H); C NMR (150 MHz, DMSO-d ) δ
74.5, 171.2, 146.6, 139.9, 139.3, 134.1, 129.5, 118.8, 112.3, 109.6, 64.1, 62.3, 36.3, 33.6, 28.4, 28.4,
+
[M+H] 393.1656, found 393.1648.
5.0, 24.4, 14.7; HRESI-MS calcd. for C19
H
25
N
O
2 7
.1.8. Synthesis of tert-butyl
(2-(6-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-6-oxohexanamido)phenyl)carbamate (6a)
To a stirred solution of 5a (92 mg, 0.25 mmol) in DMF (1.5 mL) was added tert-butyl
2-aminophenylcarbamate (53 mg, 0.25 mmol), HATU (106 mg, 0.28 mmol), and triethylamine (0.07
mL, 0.52 mmol). After stirring at room temperature for 4 h under nitrogen atmosphere, the mixture was
poured into 40 mL water. The precipitate was filtered, washed with a small amount of water.
Purification by column chromatography on silica gel (dichloromethane/methanol: 100/1) gave 85 mg
1
of 6a as an orange-solid, yield 61%, mp. 141–142 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.93 (s, 1H),
9
1
1
.46 (s, 1H), 8.32 (s, 1H), 8.02 (s, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.42–7.39 (m, 2H), 7.30 (d, J = 2.1 Hz,
H), 7.13 (td, J = 7.8, 1.2 Hz, 1H), 7.06 (td, J = 7.7, 1.4 Hz, 1H), 5.18 (s, 2H), 4.00 (t, J = 10.4 Hz, 2H),
1
3
6
.66–1.62 (m, 4H), 1.64 (s, 4H), 1.43 (s, 9H), 1.33 (t, J = 6.9 Hz, 3H); C NMR (150 MHz, DMSO-d )
δ 171.6, 171.0, 153.1, 146.6, 139.9, 139.3, 134.1, 131.1, 129.6, 129.4, 125.1, 124.9, 123.9, 123.7, 118.8,
12.2, 109.6, 79.3, 64.1, 62.3, 36.1, 35.8, 28.0, 24.9, 24.7, 14.6; HRESI-MS calcd. for C28
1
35 4 8
H N O
+
[
M+H] 555.2449, found 555.2446.
.1.9. tert-Butyl
2-(7-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-7-oxoheptanamido)phenyl)carbamate (6b)
4
(
Following the same procedures described for 6a, intermediate 6b was prepared from 5b (100 mg,
0.26 mmol), tert-butyl 2-aminophenylcarbamate (55 mg, 0.26 mmol), HATU (106 mg, 0.28 mmol), and
1
triethylamine (0.07 mL, 0.52 mmol), in 61% yield (91 mg), as an orange-red solid, mp. 138–140 °C. H
NMR (600 MHz, DMSO-d ) δ 9.90 (s, 1H), 9.43 (s, 1H), 8.33 (s, 1H), 8.02 (s, 1H), 7.52 (d, J = 7.8 Hz,
6

1H), 7.42–7.41 (m, 2H), 7.30 (d, J = 2.2 Hz, 1H), 7.12 (td, J = 7.9, 1.2 Hz, 1H), 7.05 (td, J = 7.7, 1.4
Hz, 1H), 5.18 (s, 2H), 3.99 (q, J = 7.0 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H),
1
3
1
1
7
5
4
6
.65–1.62 (m, 4H), 1.45 (s, 9H), 1.41–1.32 (m, 5H); C NMR (150 MHz, DMSO-d ) δ 171.7, 171.1,
53.1, 146.6, 139.9, 139.3, 134.1, 131.1, 129.7, 129.5, 125.0, 124.8, 123.9, 123.7, 118.8, 112.2, 109.6,
+
37 4 8
9.3, 64.1, 62.3, 40.0, 36.1, 35.8, 28.1, 25.0, 24.8, 14.6; HRESI-MS calcd. for C29H N O [M+H]
69.2606, found 569.2606.
.1.10. tert-Butyl
(2-(8-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-8-oxooctanamido)phenyl)carbamate (6c)
Following the same procedures described for 6a, intermediate 6c was prepared from 5c (100 mg,
0.25 mmol), tert-butyl 2-aminophenylcarbamate (53 mg, 0.25 mmol), HATU (106 mg, 0.28 mmol), and
1
triethylamine (0.07 mL, 0.52 mmol), in 60% yield (89 mg), as an orange-red solid, mp. 174–175 °C. H
NMR (600 MHz, DMSO-d
H), 7.41 (d, J = 2.2 Hz, 2H), 7.31 (d, J = 2.1 Hz, 1H), 7.14 (t, J = 7.0 Hz, 1H), 7.07 (td, J = 7.7, 1.4
Hz, 1H), 5.20 (s, 2H), 4.01 (q, J = 6.9 Hz, 2H), 2.35 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H),
6
) δ 9.90 (s, 1H), 9.44 (s, 1H), 8.32 (s, 1H), 8.03 (s, 1H), 7.53 (d, J = 7.7 Hz,
1
1
3
1
1
7
.64–1.59 (m, 4H), 1.46 (s, 9H), 1.36–1.32 (m, 7H); C NMR (150 MHz, DMSO-d
6
) δ 172.2, 171.6,
53.5, 147.0, 140.4, 139.8, 134.5, 131.6, 130.2, 129.9, 125.5, 125.3, 124.4, 124.2, 119.3, 112.7, 110.1,
39 4 8
9.8, 64.6, 62.8, 36.8, 36.4, 29.0, 28.8, 28.5, 25.6, 25.5, 15.1; HRESI-MS calcd. for C30H N O
+
M+H] 583.2762, found 583.2760.
[
4
.1.11. tert-Butyl
3-(6-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-6-oxohexanamido)-(1,1'-biphenyl)-4-yl)carbamat
e (7a)
(
Following the same procedures described for 6a, intermediate 7a was prepared from 5a (81 mg,
.22 mmol), tert-butyl 3-amino-4-(1,1'-biphenyl)-4-yl)carbamate [21] (63 mg, 0.22 mmol), HATU (92
0
mg, 0.28 mmol), and triethylamine (0.06 mL, 0.44 mmol), in 47% yield (65 mg), as an orange-red solid,
1
mp. 199–201 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.94 (s, 1H), 9.53 (s, 1H), 8.45 (s, 1H), 8.02 (s, 1H),
7
.74 (d, J = 1.9 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 7.3 Hz, 2H), 7.47–7.42 (m, 4H), 7.34 (t,
J = 7.3 Hz, 1H), 7.30 (d, J = 2.1 Hz, 1H), 5.18 (s, 2H), 3.99 (q, J = 6.9 Hz, 2H), 2.41 (t, J = 7.3 Hz, 2H),
1
3
2
.34 (t, J = 7.4 Hz, 2H), 1.68–1.84 (m, 4H), 1.46 (s, 9H), 1.32 (t, J = 6.9 Hz, 4H); C NMR (150 MHz,
DMSO-d ) δ 171.8, 171.0, 153.0, 146.6, 139.9, 139.4, 139.3, 135.6, 134.1, 130.5, 129.8, 129.4, 129.0,
27.3, 126.3, 123.7, 123.2, 122.9, 118.8, 112.2, 109.6, 79.5, 64.1, 62.3, 36.2, 35.9, 28.1, 24.9, 24.7,
6
1

+
1
39 4 8
4.6; HRESI-MS calcd. for C34H N O [M+H] 631.2762, found 231.2762.
4.1.12. tert-Butyl
(3-(7-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-7-oxoheptanamido)-(1,1'-biphenyl-4-yl)carbamate
(7b)
Following the same procedures described for 6a, intermediate 7b was prepared from 5b (64 mg,
0
0
1
7
.17 mmol), tert-butyl 3-amino-4-(1,1'-biphenyl)-4-yl)carbamate (48 mg, 0.17 mmol), HATU (61 mg,
.19 mmol), and triethylamine (0.05 mL, 0.33 mmol), in 64% yield (70 mg), as an orange-red solid, mp.
1
80–181 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.87 (s, 1H), 9.47 (s, 1H), 8.41 (s, 1H), 7.96 (s, 1H),
.69 (s, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.7 Hz, 3H), 7.35 (d, J = 2.2
Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 5.13 (d, J = 0.4 Hz, 2H), 3.94 (q, J = 7.0 Hz,
2
3
1
6
H), 2.35 (t, J = 7.3 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.69–1.60 (m, 4H), 1.43 (s, 9H), 1.41–1.37 (m,
1
3
H), 1.28 (t, J = 7.0 Hz, 3H); C NMR (150 MHz, DMSO-d ) δ 171.4, 171.2, 146.6, 141.4, 140.4,
6
39.9, 139.3, 134.1, 129.5, 128.9, 128.1, 126.0, 125.5, 123.9, 123.8, 123.3, 118.8, 116.2, 112.3, 109.6,
+
[M+H] 645.2919,
4.1, 62.3, 36.3, 35.8, 28.5, 25.2, 25.0, 14.7; HRESI-MS calcd. for C35
41
H N
4
O
8
found 645.2920.
.1.13. tert-Butyl
4
(3-(8-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-8-oxooctanamido)-(1,1'-biphenyl)-4-yl)carbamate
(7c)
Following the same procedures described for 6a, intermediate 7c was prepared from 5c (90 mg,
0.23 mmol), tert-butyl 3-amino-4-(1,1'-biphenyl)-4-yl)carbamate (66 mg, 0.23 mmol), HATU (96 mg,
0.28 mmol), and triethylamine (0.06 mL, 0.46 mmol), in 66% yield (101 mg), as an orange-red solid,
1
yield 66.3%, mp165–166 °C.. H NMR (600 MHz, DMSO-d
6
) δ 9.86 (s, 1H), 9.48 (s, 1H), 8.39 (s, 1H),
7.97 (s, 1H), 7.68 (d, J = 1.8 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.7
Hz, 3H), 7.35 (d, J = 2.1 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.26 (d, J = 2.1 Hz, 1H), 5.14 (s, 2H), 3.95
(q, J = 7.0 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.60–1.55 (m, 4H), 1.42 (s, 9H),
1
3
1
.32–1.31(m, 4H), 1.29 (t, J = 7.0 Hz, 3H); C NMR (150 MHz, DMSO-d
46.6, 139.9, 139.4, 139.3, 135.6, 134.1, 130.5, 129.8, 129.4, 129.0, 127.3, 126.3, 123.8, 123.2, 122.9,
18.8, 112.2, 109.6, 79.5, 64.1, 62.3, 36.2, 35.9, 28.1, 24.9, 24.7, 14.6; HRESI-MS calcd. for
6
) δ 171.8, 171.0, 153.0,
1
1
+
[M+H] 659.7480, found 659.7472.
C
36
43
H N
O
4 8
4.1.14. tert-Butyl

(
2-(6-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-6-oxohexanamido)-4-(5-methylthiophen-2-yl)phe
nyl)carbamate (8a)
Following the same procedures described for 6a, intermediate 8a was prepared from 5a (81 mg,
.22 mmol), tert-butyl 3-amino-4-(5-methylthiophen-2-yl)phenyl)carbamate [22] (68 mg, 0.22 mmol),
0
HATU (93 mg, 0.24 mmol), and triethylamine (0.06 mL, 0.44 mmol), in 57% yield (82 mg), as an
1
orange-red solid, mp. 137–139 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.91 (s, 1H), 9.48 (s, 1H), 8.42 (s,
1H), 8.01 (s, 1H), 7.66 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.40 (d, J = 1.7 Hz, 1H), 7.36 (dd, J = 8.5, 1.4
Hz, 1H), 7.31 (d, J = 1.8 Hz, 1H), 7.18 (d, J = 3.3 Hz, 1H), 6.79 (d, J = 2.8 Hz, 1H), 5.19 (s, 2H), 4.00
(q, J = 6.9 Hz, 2H), 2.45 (s, 3H), 2.39 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.68–1.62 (m, 4H),
1
3
1
1
1
6
.47 (s, 9H), 1.41–1.36 (m, 2H), 1.33 (t, J = 6.9 Hz, 3H); C NMR (150 MHz, DMSO-d ) δ 171.8,
71.0, 153.0, 146.6, 140.3, 139.9, 139.3, 138.8, 134.1, 130.0, 129.9, 129.7, 129.4, 126.8, 124.0, 123.1,
21.6, 121.1, 118.8, 112.2, 109.6, 79.5, 64.1, 62.3, 36.1, 35.8, 28.0, 24.8, 24.7, 15.1, 14.6; HRESI-MS
+
calcd. for C33
.1.15. tert-Butyl
2-(7-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-7-oxoheptanamido)-4-(5-methylthiophen-2-yl)phe
nyl)carbamate (8b)
Following the same procedures described for 6a, intermediate 8b was prepared from 5b (81 mg,
.21 mmol), tert-butyl 3-amino-4-(5-methylthiophen-2-yl)phenyl)carbamate [22] (65 mg, 0.21 mmol),
39 4
H N O
8
S [M+H] 651.2483, found 651.2479.
4
(
0
HATU (87 mg, 0.23 mmol), and triethylamine (0.06 mL, 0.44 mmol), in 58% yield (82 mg), as an
1
orange-red solid, mp. 132–133 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.92 (s, 1H), 9.48 (s, 1H), 8.42 (s,
1H), 8.01 (s, 1H), 7.65 (d, J = 1.8 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H), 7.36 (dd,
J = 8.5, 2.0 Hz, 1H), 7.30 (d, J = 2.1 Hz, 1H), 7.18 (d, J = 3.5 Hz, 1H), 6.79 (dd, J = 3.5, 1.0 Hz, 1H),
5.18 (s, 2H), 3.99 (q, J = 6.9 Hz, 2H), 2.45 (s, 3H), 2.38 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H),
1
3
1
.69–1.60 (m, 4H), 1.46 (s, 9H), 1.41–1.37 (m, 2H), 1.33 (t, J = 7.0 Hz, 3H); C NMR (150 MHz,
) δ 171.8, 171.0, 153.0, 146.6, 140.3, 139.9, 139.3, 138.8, 134.1, 130.0, 129.9, 129.7, 129.4,
26.8, 124.0, 123.1, 121.6, 121.0, 118.8, 112.2, 109.6, 79.5, 64.1, 62.3, 36.1, 35.8, 28.0, 24.8, 24.7,
DMSO-d
6
1
1
4
+
41 4 8
5.1, 14.6; HRESI-MS calcd. for C34H N O S [M+H] 665.2640, found 665.2632.
.1.16. tert-Butyl
(2-(8-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-8-oxooctanamido)-4-(5-methylthiophen-2-yl)phen
yl)carbamate (8c)

Following the same procedures described for 6a, intermediate 8c was prepared from 5c (80 mg,
.20 mmol), tert-butyl 3-amino-4-(5-methylthiophen-2-yl)phenyl)carbamate (62 mg, 0.20 mmol),
0
HATU (84 mg, 0.20 mmol), and triethylamine (0.06 mL, 0.44 mmol), in 74% yield (102 mg), as an
1
orange-red solid, mp. 153–155 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.90 (s, 1H), 9.48 (s, 1H), 8.39 (s,
1H), 8.02 (s, 1H), 7.64 (s, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 1.7 Hz, 1H), 7.36 (dd, J = 8.4, 1.8
Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.19 (d, J = 3.3 Hz, 1H), 6.79 (d, J = 2.4 Hz, 1H), 5.18 (s, 2H), 3.99
(q, J = 6.9 Hz, 2H), 2.45 (s, 3H), 2.37 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.64–1.59 (m, 4H),
1
3
1
1
8
.45 (s, 9H), 1.36–1.35 (m, 7H); C NMR (150 MHz, DMSO-d
6
) δ 172.4, 171.6, 153.5, 147.0, 140.8,
40.4, 139.7, 139.2, 134.5, 130.5, 130.2, 129.9, 127.2, 124.5, 123.6, 122.0, 121.5, 119.3, 112.7, 110.0,
43 4 8
0.0, 64.6, 62.8, 36.8, 36.4, 29.0, 28.9, 28.5, 25.5, 15.5, 15.1; HRESI-MS calcd. for C35H N O S
+
M+H] 679.2796, found 679.2790.
[
1
.1.17. Synthesis of N -(2-aminophenyl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)adipamide (9a)
6
4
To a solution of 6a (30 mg, 0.054 mmol) in DCM (1.6 mL) was added TFA (0.4 mL). After
stirring at room temperature under nitrogen atmosphere for 4 h, the mixture was concentrated under
reduced pressure. The resulting residue was dissolved in ethyl acetate, washed with saturation NaHCO
water and brine, dried over Na SO , filtered, and concentrated. Recrystallization with DCM and hexane
1/2) afforded 16 mg of 9a (65%) as an orange-yellow solid, mp. 201–203 °C. H NMR (600 MHz,
3
,
2
4
1
(
DMSO-d ) δ 9.95 (s, 1H), 9.12 (s, 1H), 8.03 (s, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H),
6
7.15 (dd, J = 7.8, 0.8 Hz, 1H), 6.88 (td, J = 7.8, 1.2 Hz, 1H), 6.71 (dd, J = 7.9, 1.0 Hz, 1H), 6.53 (td, J
=
7.8, 1.2 Hz, 1H), 5.19 (s, 2H), 4.83 (s, 2H), 4.00 (q, J = 6.9 Hz, 2H), 2.35–2.31 (m, 4H), 1.63–1.62
1
3
(
m, 4H), 1.33 (t, J = 6.9 Hz, 3H); C NMR (150 MHz, DMSO-d
6
) δ 171.1, 171.0, 146.6, 141.9, 139.9,
1
2
4
39.3, 134.1, 129.5, 125.7, 125.3, 123.5, 118.9, 116.1, 115.9, 112.3, 109.6, 64.1, 62.3, 36.2, 35.6, 25.1,
+
27 4 6
4.8, 14.7; HRESI-MS calcd. for C23H N O [M+H] 455.1925, found 455.1919.
1
.1.18. N -(2-Aminophenyl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)heptanediamide (9b)
7
Following the same procedures described for 9a, diamide 9b was prepared from 6b (80 mg, 0.14
1
mmol) and TFA (0.4 mL) in 56% yield (37 mg), as an orange-yellow solid, mp. 195–197 °C. H NMR
(
600 MHz, DMSO-d
6
) δ 9.92 (s, 1H), 9.09 (s, 1H), 8.03 (s, 1H), 7.40 (s, 1H), 7.31 (s, 1H), 7.14 (d, J =
.4 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H), 6.71 (d, J = 7.7 Hz, 1H), 6.52 (t, J = 7.3 Hz, 1H), 5.19 (s, 2H),
.81 (s, 2H), 4.00 (q, J = 6.8 Hz, 2H), 2.31 (dd, J = 15.2, 7.5 Hz, 4H), 1.66–1.61 (m, 4H), 1.39–1.34 (m,
7
4
5
1
3
H), C NMR (150 MHz, DMSO-d ) δ 171.6, 171.5, 147.0, 142.3, 140.4, 134.5, 129.9, 126.1, 125.7,
6

124.0, 119.30, 116.6, 116.3, 112.7, 110.1, 64.6, 62.8, 36.7, 36.1, 28.8, 25.6, 25.4, 15.1; HRESI-MS
+
[M+H] 469.2082, found 469.2082.
calcd. for C24
29 4
H N O
6
1
.1.19. N -(2-Aminophenyl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)octanediamide (9c)
8
4
Following the same procedures described for 9a, diamide 9c was prepared from 6c (80 mg, 0.137
1
mmol) and TFA (0.2 mL) in 51% yield (34 mg), as an orange-yellow solid, mp. 192–194 °C. H NMR
(
600 MHz, DMSO-d
.7 Hz, 1H), 6.88 (t, J = 7.1 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 6.53 (t, J = 7.5 Hz, 1H), 5.19 (s, 2H),
.80 (s, 2H), 4.00 (q, J = 6.9 Hz, 2H), 2.30 (dd, J = 15.2, 7.4 Hz, 4H), 1.62–1.58 (m, 4H), 1.35–1.32 (m,
6
) δ 9.89 (s, 1H), 9.07 (s, 1H), 8.02 (s, 1H), 7.40 (s, 1H), 7.31 (s, 1H), 7.14 (d, J =
7
4
7
1
1
3
H); C NMR (100 MHz, DMSO-d ) δ 171.6, 171.5, 147.0, 142.3, 140.4, 139.8, 134.5, 129.9, 126.1,
6
26.7, 124.1, 119.3, 116.6, 116.4, 112.8, 110.1, 64.6, 62.8, 36.8, 36.2, 29.0, 25.7, 25.5, 15.1;
+
[M+H] 483.2238, found 483.2228.
HRESI-MS calcd. for C25
31
H N
4
O
6
1
.1.20. N -(4-Amino-(1,1'-biphenyl)-3-yl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)adipamide (10a)
6
4
Following the same procedures described for 9a, diamide 10a was prepared from 7a (45 mg, 0.07
1
mmol) and TFA (0.4 mL) in 96% yield (40 mg), as an orange-yellow solid, mp. 210–212 °C. H NMR
(
600 MHz, DMSO-d
.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 2H), 7.36 (d, J = 1.6 Hz, 1H), 7.23–7.21 (m, 2H), 5.19 (s, 2H), 5.10 (s,
H), 4.00 (q, J = 6.9 Hz, 2H), 2.41 (t, J = 6.3 Hz, 2H), 2.36 (t, J = 6.4 Hz, 2H), 1.66 (s, 4H), 1.33 (t, J =
6
) δ 10.08 (s, 1H), 9.33 (s, 1H), 8.02 (s, 1H), 7.57 (d, J = 1.7 Hz, 1H), 7.45 (d, J =
1
2
6
1
2
4
1
3
.9 Hz, 3H); C NMR (150 MHz, DMSO-d ) δ 171.3, 171.2, 146.5, 141.3, 140.4, 139.9, 139.3, 134.2,
6
29.5, 128.8, 127.9, 125.9, 125.5, 124.0, 123.7, 123.1, 118.8, 116.2, 112.3, 109.7, 64.1, 62.3, 36.0, 35.5,
+
[M+H] 531.2238, found 531.2240.
4.9, 14.7; HRESI-MS calcd. for C29
31
H N
4
O
6
1
N -(4-Amino-(1,1'-biphenyl)-3-yl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)heptanediamide
7
.1.21.
10b)
(
Following the same procedures described for 9a, diamide 10b was prepared from 7b (50 mg, 0.08
1
mmol) and TFA (0.4 mL) in 96% yield (45 mg), as an orange-yellow solid, mp. 181–183 °C. H NMR
(
600 MHz, DMSO-d
6
) δ 9.96 (s, 1H), 9.22 (s, 1H), 8.00 (s, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.50 (d, J =
.4 Hz, 2H), 7.41 (d, J = 1.9 Hz, 1H), 7.37 (t, J = 7.7 Hz, 2H), 7.32 (d, J = 2.0 Hz, 1H), 7.23–7.21 (m,
H), 6.79 (d, J = 8.3 Hz, 1H), 5.17 (s, 2H), 5.04 (s, 2H), 3.99 (q, J = 6.9 Hz, 2H), 2.36 (t, J = 7.4 Hz,
7
2
2
1
H), 2.31 (t, J = 7.4 Hz, 2H), 1.66–1.61 (m, 4H), 1.39–1.34 (m, 2H), 1.32 (t, J = 6.9 Hz, 3H). C NMR
3
(150 MHz, DMSO-d
6
) δ 171.4, 171.2, 146.6, 141.4, 140.4, 139.9, 139.3, 134.2, 129.5, 128.8, 128.0,

1
1
4
25.9, 125.5, 123.9, 123.8, 123.2, 118.8, 116.2, 112.3, 109.7, 64.1, 62.3, 36.2, 35.7, 28.3, 25.1, 24.9,
+
[M+H] 545.2395, found 545.2395.
4.7; HRESI-MS calcd. for C30
H
33
4
N O
6
1
N -(4-Amino-(1,1'-biphenyl)-3-yl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)octanediamide
8
.1.22.
(10c)
Following the same procedures described for 9a, diamide 10c was prepared from 7c (80 mg, 0.12
1
mmol) and TFA (0.4 mL) in 94% yield (71 mg), as an orange-yellow solid, mp. 172–173°C. H NMR
(
600 MHz, DMSO-d
6
) δ 9.91 (s, 1H), 9.17 (s, 1H), 8.01 (s, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.50 (d, J =
.4 Hz, 2H), 7.40 (d, J = 2.0 Hz, 1H), 7.37 (t, J = 7.7 Hz, 2H), 7.31 (d, J = 1.9 Hz, 1H), 7.24–7.20 (m,
H), 6.79 (d, J = 8.3 Hz, 1H), 5.17 (s, 2H), 5.02 (s, 2H), 3.99 (q, J = 6.9 Hz, 2H), 2.34 (t, J = 7.4 Hz,
7
2
2
13
6
H), 2.28 (t, J = 7.3 Hz, 2H), 1.63–1.58 (m, 4H), 1.35–1.30 (m, 7H); C NMR (150 MHz, DMSO-d )
δ 171.8, 171.7, 147.0, 141.9, 140.8, 140.3, 139.7, 134.6, 129.9, 129.2, 128.5, 126.4, 126.0, 124.4, 124.3,
123.7, 119.3, 116.7, 112.7, 110.1, 64.6, 62.8, 36.8, 36.8, 29.0, 25.7, 25.5, 15.1; HRESI-MS calcd. for
+
[M+H] 559.2551, found 559.2543.
C
31
35
H N
O
4 6
4.1.23.
1
N -(2-Amino-5-(5-methylthiophen-2-yl)phenyl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)adipamide
6
(11a)
Following the same procedures described for 9a, diamide 11a was prepared from 8a (40 mg, 0.06
1
mmol) and TFA (0.2 mL) in 89% yield (30 mg), as a yellow solid, mp. 214–215 °C. H NMR (600
MHz, DMSO-d ) δ 9.93 (s, 1H), 9.15 (s, 1H), 8.02 (s, 1H), 7.40 (t, J = 2.0 Hz, 2H), 7.30 (d, J = 2.1 Hz,
H), 7.12 (dd, J = 8.2, 2.2 Hz, 1H), 6.95 (d, J = 3.5 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 1.2
Hz, 1H), 5.17 (s, 2H), 5.03 (s, 2H), 3.99 (q, J = 6.9 Hz, 3H), 2.39 (s, 3H), 2.35–2.32 (m, 4H),
6
1
1
3
1
1
1
5
4
6
.65–1.61 (m, 4H), 1.32 (t, J = 6.9 Hz, 4H); C NMR (100 MHz, DMSO-d ) δ 171.2, 171.1, 146.6,
42.0, 141.3, 139.9, 139.3, 136.4, 134.0, 129.5, 126.3, 123.6, 122.7, 122.5, 122.0, 120.6, 118.8, 116.1,
+
31 4 6
12.3, 109.7, 64.1, 62.3, 36.2, 35.6, 25.0, 24.8, 15.0, 14.6; HRESI-MS calcd. for C28H N O S [M+H]
51.1959, found 551.1961.
.1.24.
1
N -(2-Amino-5-(5-methylthiophen-2-yl)phenyl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)heptanediam
7
ide (11b)
Following the same procedures described for 9a, diamide 11b was prepared from 8b (42 mg, 0.06
1
mmol) and TFA (0.4 mL) in 88% yield (30 mg), as a yellow solid, mp. 163–165 °C. H NMR (600

MHz, DMSO-d
6
) δ 9.91 (s, 1H), 9.13 (s, 1H), 8.01 (s, 1H), 7.40 (dd, J = 6.8, 2.1 Hz, 2H), 7.30 (d, J =
2.1 Hz, 1H), 7.12 (dd, J = 8.3, 2.1 Hz, 1H), 6.96 (d, J = 3.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.69 (d, J
=
1.1 Hz, 1H), 5.18 (s, 2H), 5.02 (s, 2H), 3.99 (dd, J = 13.6, 6.6 Hz, 2H), 2.40 (s, 3H), 2.36–2.26 (m,
1
3
4
1
1
H), 1.66–1.59 (m, 4H), 1.39–1.35 (m, 5H); C NMR (150 MHz, DMSO-d
6
) δ 171.3, 171.1, 146.6,
42.0, 141.3, 139.9, 139.3, 136.4, 134.1, 129.5, 126.3, 123.7, 122.7, 122.5, 121.9, 120.6, 118.8, 116.08,
33 4 6
12.3, 109.7, 64.1, 62.3, 36.2, 35.6, 28.3, 25.0, 24.9, 15.0, 14.6; HRESI-MS calcd. for C29H N O S
+
M+H] 565.2115, found 565.2110.
[
4.1.25.
1
N -(2-Amino-5-(5-methylthiophen-2-yl)phenyl)-N -(8-ethoxy-3-nitro-2H-chromen-6-yl)octanediami
8
de (11c)
Following the same procedures described for 9a, diamide 11c was prepared from 8c (80 mg, 0.12
1
mmol) and TFA (0.4 mL) in 88% yield (60 mg), as a yellow solid, mp. 164–166 °C. H NMR (600
MHz, DMSO-d
.1 Hz, 1H), 7.13 (dd, J = 8.3, 2.1 Hz, 1H), 6.96 (d, J = 3.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.70 (dd,
J = 3.4, 1.1 Hz, 1H), 5.18 (s, 2H), 5.02 (s, 2H), 3.99 (q, J = 7.0 Hz, 2H), 2.40 (s, 3H), 2.33 (t, J = 7.4
6
) δ 9.90 (s, 1H), 9.14 (s, 1H), 8.02 (s, 1H), 7.40 (dd, J = 7.9, 2.1 Hz, 2H), 7.31 (d, J =
2
1
3
Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.63–1.59 (m, 4H), 1.36–1.35 (m, 7H); C NMR (150 MHz,
DMSO-d ) δ 171.3, 171.2, 146.6, 142.0, 141.3, 139.9, 139.3, 136.4, 134.1, 129.5, 126.4, 123.7, 122.7,
22.5, 122.0, 120.6, 118.8, 116.1, 112.3, 109.7, 64.1, 62.3, 36.3, 35.8, 28.5, 25.1, 25.0, 24.9, 15.0, 14.6;
6
1
+
S [M+H] 579.2272, found 579.2260.
HRESI-MS calcd. for C30
35
H N
4
O
6
1
.1.26. Synthesis of N -(8-ethoxy-3-nitro-2H-chromen-6-yl)-N -phenethyladipamide (12a)
6
4
To a solution of 5a (60 mg, 0.16 mmol) and HATU (68 mg, 0.18 mmol) in anhydrous DMF (2 mL)
was added triethylamine (0.05 mL, 0.32 mmol). After stirring for 30 min at room temperature under
nitrogen atmosphere, phenylethylamine (0.021 mL, 0.16 mmol) was added. The resulting mixture was
stirred for another 4 h. The mixture was poured into 20 mL water and filtered. The precipitate was
dissolved in ethyl acetate and filtered, and the filtrate was concentrated under reduced pressure.
Recrystallization with EtOAc and hexane (1/2) afforded 50 mg of 12a (67%) as a orange-red solid, mp.
1
33–234 °C. H NMR (600 MHz, DMSO-d
2
6
) δ 9.89 (s, 1H), 8.01 (s, 1H), 7.87 (t, J = 5.1 Hz, 1H), 7.39
(d, J = 1.4 Hz, 1H), 7.29 (d, J = 1.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 2H), 7.18–7.14 (m, 3H), 5.17 (s, 2H),
3.99 (q, J = 6.9 Hz, 2H), 3.24 (dd, J = 13.3, 6.8 Hz, 2H), 2.67 (t, J = 7.3 Hz, 2H), 2.25 (t, J = 6.6 Hz,
1
3
2
H), 2.05 (t, J = 6.6 Hz, 2H), 1.51–1.48 (m, 4H), 1.32 (t, J = 6.9 Hz, 3H); C NMR (150 MHz,

DMSO-d ) δ 171.9, 171.1, 146.6, 139.9, 139.5, 139.3, 134.1, 129.5, 128.7, 128.3, 126.1, 118.9, 112.2,
6
+
1
4
4
09.9, 64.1, 62.3, 40.1, 36.2, 35.3, 35.2, 25.1, 24.9, 14.7; HRESI-MS calcd. for C25
30 3 6
H N O [M+H]
68.2129, found 468.2120.
1
.1.27. N -(8-Ethoxy-3-nitro-2H-chromen-6-yl)-N -phenethylheptanediamide (12b)
7
Following the same procedures described for 12a, diamide 12b was prepared from 5b (78 mg,
0.21 mmol), phenylethylamine (0.026 mL, 0.21 mmol), HATU (87 mg, 0.21 mmol), and triethylamine
1
0.06 mL, 0.44 mmol), in 62% yield (60 mg), as an orange-red solid, mp. 180–182 °C. H NMR (600
(
MHz, DMSO-d ) δ 9.87 (s, 1H), 7.99 (s, 1H), 7.84 (s, 1H), 7.38 (s, 1H), 7.28 (s, 1H), 7.25 (t, J = 7.4
6
Hz, 2H), 7.16 (d, J = 7.3 Hz, 3H), 5.16 (s, 2H), 3.97 (dd, J = 13.6, 6.7 Hz, 2H), 3.22 (dd, J = 13.1, 6.6
Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.24 (t, J = 7.1 Hz, 2H), 2.01 (t, J = 7.1 Hz, 2H), 1.56–1.51 (m, 2H),
1
3
1
1
6
6
.49 – 1.44 ( m, 2H), 1.30 (t, J = 6.8 Hz, 3H), 1.24–1.19 (m, 2H); C NMR (150 MHz, DMSO-d ) δ
72.2, 171.4, 146.8, 140.1, 139.7, 139.5, 134.3, 129.7, 128.8, 128.5, 126.2, 119.0, 112.4, 109.8, 64.3,
+
[M+H] 482.2286,
2.5, 40.3, 36.4, 35.5, 35.4, 28.5, 25.3, 25.1, 14.8; HRESI-MS calcd. for C26
H
32
N
O
3 6
found 482.2278.
1
.1.28. N -(8-Ethoxy-3-nitro-2H-chromen-6-yl)-N -phenethyloctanediamide (12c)
8
4
Following the same procedures described for 12a, diamide 12c was prepared from 5c (57 mg, 0.15
mmol), phenylethylamine (0.018 mL, 0.15 mmol), HATU (65 mg, 0.17 mmol), and triethylamine (0.04
1
mL, 0.29 mmol), in 65% yield (47 mg), as an orange-red solid, mp. 211–212 °C. H NMR (600 MHz,
DMSO-d
.19 (d, J = 7.0 Hz, 3H), 5.18 (s, 2H), 4.00 (q, J = 6.8 Hz, 2H), 3.24 (dd, J = 13.1, 6.7 Hz, 2H), 2.68 (t,
J = 7.2 Hz, 2H), 2.50 (s, 3H), 2.26 (s, 2H), 2.02 (t, J = 7.2 Hz, 2H), 1.55 (dd, J = 14.1, 7.0 Hz, 2H),
6
) δ 9.89 (s, 1H), 8.02 (s, 1H), 7.84 (s, 1H), 7.40 (s, 1H), 7.31 (s, 1H), 7.27 (t, J = 7.5 Hz, 2H),
7
1
3
1
1
6
4
4
6
.47–1.44 (m, 2H), 1.33 (t, J = 6.9 Hz, 3H), 1.27–1.22 (m, 4H); C NMR (150 MHz, DMSO-d ) δ
72.0, 171.2, 146.6, 139.9, 139.6, 139.3, 134.1, 129.5, 128.6, 128.3, 126.0, 118.9, 112.3, 109.6, 64.1,
+
2.3, 40.1, 36.3, 35.4, 35.2, 28.5, 28.4, 25.2, 25.0, 14.7; HRESI-MS calcd. for C27
96.2442, found 496.2442.
34 3 6
H N O [M+H]
.1.29. Synthesis of tert-butyl (4-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-4-oxobutyl)carbamate
13a)
To a solution of 4 (100 mg, 0.30 mmol) in DCM (1.6 mL) was added TFA (0.4 mL). After stirring
(
at room temperature for 2 h, the mixture was concentrated under reduced pressure. The resulting
residue was dissolved in anhydrous DMF (2 mL), 5-(tert-butoxycarbonylamino)pentanoic acid (71 mg,

0.33 mmol), HATU (125 mg, 0.33 mmol) and triethylamine (0.17 mL, 1.2 mmol) were added. After
stirring for 12 h, the mixture was poured into 40 mL water. The precipitate was filtered and dried to
1
give 120 mg of 13a (92%) as a yellow solid, mp. 199–200 °C. H NMR (600 MHz, CDCl
3
) δ 7.69 (s,
1
2
1
1
H), 7.66 (s, 1H), 7.35 (d, J = 1.3 Hz, 1H), 6.99 (s, 1H), 5.22 (s, 2H), 4.65 (s, 1H), 4.06 (q, J = 7.0 Hz,
H), 3.15 (dd, J = 12.3, 6.0 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 1.77–1.72 (m, 2H), 1.56–1.53 (m, 3H),
1
3
.41 (t, 3H), 1.40 (s, 9H); C NMR (150 MHz, CDCl ) δ 171.6, 156.7, 147.5, 140.5, 139.7, 133.2,
3
29.2, 118.8, 112.5, 110.2, 79.5, 64.9, 63.1, 39.1, 36.6, 29.5, 28.5, 22.5, 14.7; HRESI-MS calcd. for
+
[M+H] 436.2078, found 436.2076.
C
21
30
H N
O
3 7
4
.1.30. tert-Butyl (5-((8-ethoxy-3-nitro-2H-chromen-6-yl)amino)-5-oxopentyl)carbamate (13b)
Following the same procedures described for 13a, intermediate 13b was prepared from 4 (131 mg,
.39 mmol), TFA (0.4 mL); 5-(tert-butoxycarbonylamino)hexanoic acid (100 mg, 0.43 mmol), HATU
0
(164 mg, 0.43 mmol) and triethylamine (0.22 mL, 1.56 mmol), in 89% yield (155 mg), as an
1
orange-yellow solid, mp. 160–161 °C. H NMR (600 MHz, CDCl
3
) δ 7.70 (s, 1H), 7.48 (s, 1H), 7.39 (d,
J = 1.9 Hz, 1H), 7.00 (s, 1H), 5.26 (s, 2H), 4.60 (s, 1H), 4.10 (q, J = 7.0 Hz, 2H), 3.13 (d, J = 6.4 Hz,
2H), 2.35 (t, J = 7.5 Hz, 2H), 1.76–1.73 (m, 2H), 1.55–1.51 (m, 2H), 1.46 (t, J = 7.5 Hz, 3H), 1.44 (s,
1
H), 1.42–1.36 (m, 2H); C NMR (150 MHz, CDCl
3
9
3
) δ 171.5, 156.2, 147.5, 140.6, 139.7, 133.1, 129.2,
118.8, 112.5, 110.2, 79.3, 65.0, 63.1, 40.1, 37.3, 29.6, 28.4, 26.1, 24.9, 14.7; HRESI-MS calcd. for
+
C
22
32
H N
O
3 7
[M+H] 450.2235, found 450.2227.
.1.31. Synthesis of N-(8-ethoxy-3-nitro-2H-chromen-6-yl)-6-(2-phenylacetamido)hexanamide (14)
To a solution of 13b (100 mg, 0.22 mmol) in CH Cl (1.6 mL) was added TFA (0.4 mL). After
4
2
2
stirring at room temperature for 2 h, the mixture was concentrated under reduced pressure. The
resulting residue was dissolved in anhydrous DMF (1.5 mL). Phenylacetic acid (36 mg, 0.27 mmol),
anhydrous triethylamine (0.16 mL, 1.11 mmol) and HATU (101 mg, 0.27 mmol) were added to the
mixture. After stirring for 8 h, the reaction mixture was poured into 20 mL water. The precipitate was
filtered and dried. Recrystallization with CH
2
Cl
2
and hexane (1/2) provided 99 mg of 14 as an
orange-red solid, yield 95%, mp. 220–222 °C. H NMR (600 MHz, DMSO-d ) δ 9.88 (s, 1H), 8.01 (s,
H), 7.39 (d, J = 1.5 Hz, 1H), 7.28 (d, J = 1.6 Hz, 1H), 7.26 (t, J = 7.4 Hz, 2H), 7.22 (d, J = 7.1 Hz,
H), 7.18 (t, J = 7.1 Hz, 1H), 5.17 (s, 2H), 3.98 (q, J = 6.9 Hz, 2H), 3.35 (s, 2H), 3.02 (dd, J = 12.7, 6.5
1
6
2
2
Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.57–1.52 (m, 2H), 1.42–1.37 (m, 2H), 1.31 (t, J = 6.9 Hz, 3H),
1
3
1
6
.28–1.23 (m, 2H); C NMR (150 MHz, DMSO-d ) δ 171.1, 169.9, 146.6, 139.9, 139.3, 136.6, 134.1,

129.5, 128.9, 128.2, 126.3, 118.9, 112.3, 109.6, 64.1, 42.5, 38.5, 36.2, 28.9, 26.1, 24.8, 14.7;
+
[M+H] 468.2129, found 468.2121.
HRESI-MS calcd. for C25
30
H N
3
O
6
4
.1.32. N-(8-Ethoxy-3-nitro-2H-chromen-6-yl)-6-(3-phenylpropanamido)hexanamide (15)
Following the same procedures described for 14, phenylpropanamide 15 was prepared from 13b
100 mg, 0.22 mmol), TFA (0.4 mL); 3-phenylpropanoic acid (40 mg, 0.27 mmol), HATU (101 mg,
(
0.27 mmol) and triethylamine (0.16 mL, 1.11 mmol), in 93% yield (100 mg), as an orange-yellow solid,
1
mp. 189–190 °C. H NMR (600 MHz, DMSO-d
6
) δ 9.88 (s, 1H), 8.00 (s, 1H), 7.77 (t, J = 5.3 Hz, 1H),
7
.38 (d, J = 1.9 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 7.24 (t, J = 7.5 Hz, 2H), 7.16–7.13 (m, 3H), 5.16 (s,
H), 3.98 (q, J = 6.9 Hz, 2H), 3.00 (dd, J = 12.8, 6.6 Hz, 2H), 2.77 (t, J = 7.8 Hz, 2H), 2.32 (t, J = 7.8
2
Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.56–1.51 (m, 2H), 1.38–1.34 (m, 2H), 1.31 (t, J = 6.9 Hz, 3H),
1
3
1
6
.24–1.19 (m, 2H); C NMR (150 MHz, DMSO-d ) δ 171.2, 171.1, 146.6, 141.4, 139.9, 139.3, 134.1,
129.5, 128.3, 128.2, 125.9, 118.9, 112.3, 109.6, 64.1, 62.3, 38.3, 37.1, 36.2, 31.2, 29.0, 26.1, 24.8, 14.7;
+
[M+H] 482.2286, found 482.2278.
HRESI-MS calcd. for C26
.1.33. Synthesis of
32
H N
3
O
6
4
(R)-5-(2-amino-3-phenylpropanamido)-N-(8-ethoxy-3-nitro-2H-chromen-6-yl)pentanamide (16a)
To a solution of 13a (100 mg, 0.23 mmol) in DCM (1.6 mL) was added TFA (0.4 mL). After
stirring for 2 h at room temperature, the mixture was concentrated under reduced pressure. The
resulting residue was dissolved in anhydrous DMF (1.5 mL). D-Boc-Phe (74 mg, 0.28 mmol),
triethylamine (0.15 mL, 1.11 mmoland HATU (106 mg, 0.28 mmol) were added to the mixture. After
stirring for 8 h at room temperature, the resulting mixture was poured into 20 mL water. After filtration,
the precipitate was dried and then dissolved in a 20% TFA in CH
the reaction mixture was diluted with CH Cl . The organic phase was washed with saturation NaHCO
distilled water, brine, and dried over Na SO , filtered and concentrated under reduced pressure.
2 2
Cl (2.0 mL). After stirring for 2 h,
2
2
3
,
2
4
Recrystallization with DCM and hexane (1/2) afforded 40 mg of 16a as an orange-yellow solid, yield
1
1%, mp 191–192 °C. H NMR (600 MHz, DMSO-d
5
2
3
2
1
6
6
) δ 9.92 (s, 1H), 8.29 (t, J = 5.6 Hz, 1H), 8.08 (s,
H), 8.00 (s, 1H), 7.39 (s, 1H), 7.31–7.28 (m, 3H), 7.22 (t, J = 7.4 Hz, 1H), 7.18 (s, 2H), 5.17 (s, 2H),
.97 (q, J = 7.0 Hz, 2H), 3.87 (t, J = 7.2 Hz, 1H), 3.12 (td, J = 13.2, 6.6 Hz, 1H), 3.00–2.92 (m, 3H),
13
.23 (t, J = 7.3 Hz, 2H), 1.47–1.41 (m, 2H), 1.34–1.26 (m, 5H); C NMR (150 MHz, DMSO-d ) δ
6
71.0, 167.7, 146.6, 140.0, 139.3, 135.0, 134.1, 129.4, 128.5, 127.1, 118.9, 116.4, 112.2, 109.5, 64.1,
+
[M+H] 483.2238,
2.4, 53.7, 38.4, 37.2, 35.8, 28.3, 22.4, 14.7; HRESI-MS calcd. for C25
31
H N
4
O
6

found 483.2236.
.1.34. (R)-6-(2-Amino-3-phenylpropanamido)-N-(8-ethoxy-3-nitro-2H-chromen-6-yl)hexanamide
16b)
Following the same procedures described for 16a, 6-hexanamide 16b was prepared from 13b (100
mg, 0.22 mmol), 20% TFA in CH Cl (2.0 mL); D-Boc-Phe (72 mg, 0.27 mmol), HATU (101 mg, 0.27
mmol) and triethylamine (0.15 mL, 1.11 mmol); 20% TFA in CH Cl (2.0 mL), in 48% yield (37 mg),
as an orange-yellow solid, mp. 113–115 °C. H NMR (600 MHz, DMSO-d ) δ 9.90 (s, 1H), 8.18 (t, J =
.4 Hz, 1H), 7.98 (s, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.32–7.30 (m, 3H), 7.23 (t, J = 7.3 Hz, 1H), 7.18 (d,
J = 7.2 Hz, 2H), 5.16 (s, 2H), 3.97 (q, J = 6.9 Hz, 2H), 3.77 (t, J = 7.0 Hz, 1H), 3.15–3.09 (m, 1H),
4
(
2
2
2
2
1
6
5
2
1
1
.96–2.93 (m, 2H), 2.91–2.87 (m, 1H), 2.23 (t, J = 7.3 Hz, 2H), 1.55–1.50 (m, 2H), 1.31–1.28 (m, 5H),
1
3
.19–1.14 (m, 2H); C NMR (150 MHz, DMSO-d ) δ 171.1, 168.6, 146.6, 139.9, 139.3, 135.6, 134.1,
6
29.5, 129.4, 128.5, 127.0, 118.9, 112.2, 109.6, 64.1, 62.3, 54.1, 38.5, 37.8, 36.2, 28.6, 26.0 24.7, 14.7;
+
HRESI-MS calcd. for C26
.2. In vitro antiproliferative assay
The 8-ethoxy-3-nitro-2H-chromene derivatives were subjected to in vitro growth inhibitory assay
33
H N
4
O
6
[M+H] 497.2395, found 497.2387.
4
against A549, MCF-7, K562, HeLa and PC3 cells by conventional MTT method [23]. The values of
GI50, the effective concentration at which 50% of the tumor cells were inhibited, were calculated by the
3
GraphPad Prism 5 software. Tumor cells were seeded in 96-well plates at density of 4 × 10 cells per
well. Following the overnight adherence, cells were incubated with medium alone or with the
8-ethoxy-3-nitro-2H-chromene derivatives or the reference drugs at the indicated concentrations for 48
h at 37 °C. Cells were treated with the MTT solution (final concentration, 0.5 mg/mL) for 4 h at 37 °C.
The supernatants were removed carefully, followed by the addition of 100 µL DMSO to each well to
dissolve the precipitate. The absorbance was measured at 570 nm on a microplate reader. Triplicate
experiments were performed.
4.3. In vitro HDACs inhibition assay
In vitro HDACs inhibition assays were conducted as previously described [24]. In brief, 10 µL of
enzyme solution (HDAC1, HDAC2, or HDAC6) was mixed with different concentrations of
-ethoxy-3-nitro-2H-chromene derivatives (50 µL), positive drug control SAHA and MS275, using 100%
8
and none HDACs groups as control group, and the mixture. After incubation at 37 °C for 10 min,
fluorogenic substrate Ac-Leu-GlyLys(Ac)-AMC (40 µL) was added and then the mixture was

incubated at 37 °C for 30 min. The reaction was quenched with 100 µL of stop buffer containing
trypsin and TSA. After incubation at 37 °C for 20 min, fluorescence intensity was measured using a
microplate reader at excitation and emission wavelengths of 390 and 460 nm, respectively. The
inhibition ratios were calculated from the fluorescence intensity readings of tested wells relative to
those of control wells. In the IC50 determination, each compound was tested at 8 concentrations
starting from 10 µM with 3-fold-dilution in singlet. The IC50 values were calculated using a
regression analysis of the concentration/inhibition data.
Acknowledgments
This work was partially supported by the Major Project of Science and Technology of Shandong
Province (2015ZDJS04001) and the Fundamental Research Funds of Shandong University
(2016JC025).
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NH2
H
N
H
N
O N
2
( )n
NO2
O
O
O
OEt
R
O
9
a–c; 10a–c; 11a–c
OEt
F
H
N
H
N
S14161
O N
2
( )n
Bn
O
O
O
O
Br
NO2
OEt
12a–c
O
H
N
O
O N
2
( )5
R
N
H
OEt
O
BENC-511
OEt
OEt
1
4; 15
O
H
N
Cap group
Linker
Zinc binding group (ZBG)
Internal cavity motif
O N
2
( )n
N
Ph
H
O
NH2
O
1
6a; 16b